10.2478/abm-2010-0126

Asian Biomedicine Vol. 4 No. 6 December 2010; 955-958 Brief communication (Original)

Adrenomedullin administration alters vascular endothelial growth factor levels in rats in cold stress

Nuran Cikcikoglu Yildirima, Muhittin Yureklib aDepartment of Environmental, Faculty of Engineering, Tunceli University, Tunceli 62000, Turkey; bInonu University, Faculty of Arts and Science, Department of Molecular Biology, Malatya 44280, Turkey

Background: Many endogenous peptides play important regulatory roles in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AdM) is one of such factors. Angiogenesis and vascular endothelial growth factor (VEGF) are indistinguishable. Exposure to cold environment stimulates capillary angiogenesis. Objectives: Examine the effect of the bioactive peptide AdM on VEGF levels in rat liver, lung, brain, and heart tissues after cold stress treatment. Methods: Male wistar rats were divided into four groups as control, AdM treatment, cold stress and AdM+cold stress treated groups. In AdM-treated group, animals received intraperitoneal injection of AdM (2000 ng/kg body weight) once a day during a week. For the cold stress exposure, the rats were kept in separate cages at 10°C for a week. Results: The administration of AdM increased VEGF levels in all tissues in cold exposed rats. Conclusion: AdM may be a major regulatory factor in angiogenesis by modulating VEGF levels that is closely associated with cold exposure-related metabolic stimulation.

Keywords: Adrenomedullin, angiogenesis, cold stress, rat, vascular endothelial growth factor (VEGF)

Angiogenesis, or the formation of new blood of the endothelial cells to form a capillary lumen. It is vessels from pre-existing ones [1], plays a pivotal followed by inhibition of EC proliferation and role during embrional development and later, in adult stabilization of the new vessel by basement membrane life, in a variety of physiological and pathological reconstitution and recruitment of periendothelial cells. conditions, such as malignancy and chronic Modulation of any one of these steps would affect inflammation [2]. Diseased or injured cells in response the formation of new vessels [5]. to genetic alterations, hypoxia, hypoglycemia, EC properties are regulated by many trophic mechanical stress, and/or inflammatory proteins, factors [6], such as fibroblast growth factor-2 (FGF- release pro-angiogenic growth factors such as 2) and transforming growth factors (TGFs). However, vascular endothelial growth factor (VEGF) into the VEGF is the major endogenous regulator [7] of surrounding tissue. The released growth factors bind EC proliferation, migration, and differentiation. to endothelial cells nearby blood vessels and stimulate Angiogenesis and VEGF are indistinguishable. VEGF their proliferation [3]. The angiogenic cascade is a is the potent and critical vascular regulator required complex multistep process [4]. It includes sequential to initiate the formation of immature vessels by basement membrane degradation, endothelial cell angiogenic sprouting. Inappropriate induction of VEGF, (EC) migration and proliferation, and self-organization in the absence of the entire angiogenic program, leads to formation of immature and leaky vessels that cause disease [8-10]. VEGF acts as a survival factor for Correspondance to: Nuran Cikcikoglu Yildirim, PhD, Department of Environmental Engineering, Faculty of Engineering, Tunceli VEGF, thereby protecting tumor cells from apoptosis University, Tunceli 62000 , Turkey. E-mail: [email protected] and/or necrosis [11]. 956 N. Cikcikoglu Yildirim, M. Yurekli

Exposure to cold environment and/or hypoxia Experimental design induces an increase in wall tension and shear stress At the end of the experiment, the rats were in capillary, and stimulates capillary angiogenesis [12]. anaesthetized by intraperitoneal injection of ketamine/ An increase in the number and density of capillaries xylazine hydrochloride (20/2 mg kg body weight), and may help improve the oxygen supply to muscle tissues the liver was removed immediately. Liver, lung, brain during cold exposure [13-17]. and heart tissues were homogenized (PCV Kinematica In recent years, evidence has accumulated to Status Homogenizator, Littau-Luzern, Switzerland; indicate that many endogenous peptides play important Bronson sonifier 450, Danburg, USA) in ice-cold regulatory roles in angiogenesis by modulating phosphate buffered saline (pH 7.4). The homogenate endothelial cell behavior. Adrenomedullin (AdM) is was sonified with an ultrasonifier (Bronson sonifier one of such factors [18]. It was shown that AdM 450) using six cycles (20 second sonications and induced proliferation, migration, and tube formation in 40 second pause on ice). The homogenate was ECs in vitro. It was also confirmed that AdM induced centrifuged (15000 g, 10 minutes, 4°C), and the sprouting by ECs. AdM promoted endothelial DNA supernatant was subjected to enzyme assays synthesis, migration, and tube formation, which are immediately. the essential steps for angiogenesis. Furthermore, AdM The animal experiments were performed in promoted sprouting in vitro and neovessel formation accordance with the guidelines for animal Research in vivo in gel plugs. Thus, it was a demonstrated novel from the National Institute of Health and were biological function of AdM in angiogenesis [19]. approved by the Committee of Animal Research at Truly, cold stress may stimulate capillary Inonu University, Malatya. angiogenesis [12]. However, the mechanism of angiogenesis due to cold stress is still unknown. ELISA measurement Furthermore, there is no data available on the effects VEGF levels were assayed by an enzyme-linked of prolonged cold stress on the VEGF levels in liver, immunosorbent assay (ELISA) kit (The Cayman lung, brain, and heart tissues. In the present study, we Chemical VEGF Assay kit, Michigan, USA). examined whether AdM affects the levels of VEGF in cold stress-exposed rats. Statistical analysis Statistical analysis was carried out using the SPSS Materials and methods 12.0 statistical program. All data were expressed as Treatment of animals mean±standard errors (SE). Duncan test was used to Rats were obtained from Experimental Animal determine the differences between the groups having Research Facility, Inonu University. Twenty-four male two parts. One-way analysis of variance was used to Wistar albino rats (eight months old, 190-240 g) were determine the differences between the groups having housed individually under diurnal lighting conditions more than two parts. (12-12 hour) with free access to drinking water and a standard pellet diet. The environment in the animal Results rooms was maintained at a temperature of 22±2°C, In the cold stress group, the effects of cold stress relative humidity of 45±5%. The rats were divided on VEGF levels were investigated in each tissue, into four experimental groups: control group (n = 6), which are summarized in Table 1. adrenomedullin group (AdM) (n = 6), cold stress group VEGF levels significantly increased in lung and (n = 6), and cold stress+adrenomedullin group brain tissues (p >0.05) but decreased in liver and heart (AdM+cold stress) (n = 6). The AdM-treated groups tissues, compared to the control (p <0.05). Depending received an i.p. injection of AdM (2000 ng/g body on AdM treatment, VEGF levels increased in liver weight) for a week. In cold stress treatment group, (p <0.05) and lung (p >0.05) tissues but decreased animals were exposed +10°C cold during a week. In in brain and heart tissue, compared to the control cold stress+AdM treatment groups, both animals were (p <0.05). In AdM+cold stress group, VEGF levels exposed +10°C cold during a week and were given increased in all tissues compared to control (p <0.05). AdM intraperitoneally at a single dose of 2000 ng/g Interestingly, AdM application in addition to cold stress body weight for a week. increase VEGF levels in liver, lung, brain, and heart tissues, compared to AdM treatment group (p <0.05). Vol. 4 No. 6 Adrenomedull n administriation alters VEGF levels 957 December 2010

Table 1. The effect of AdM treatment and cold stress on VEGF levels in rat liver, lung, brain, and heart tissues.

VEGF Levels (pg/mL) Groups Liver Lung Brain Heart

Control 326±12,81bc 259±17,13c 750±47,3a 429±42,95b AdM 392±3,65b 325±18,46c 458±37,98b 332±18,13c Stress 219±20,6c 327±27,08c 888±73,3a 369±11,98bc Stress+AdM 536±46,42a 649±34,19b 775±17,86a 583±13,77a

*Values shown are mean±SE for six replicate experiments. Different letters (a,b, and c) in each column indicate significant difference between groups according to Duncan’s Multiple Range Test, (p <0.05)

Discussion It has been reported that specific conditions (such The increased shear stress associated with as hypoxia) associated to increased VEGF expression elevated flow and blood viscosity in the cold, as and angiogenesis stimulate AdM synthesis and indicated by the reduction in capillary blood transit secretion leading to a strong increase of the peptide times, has been shown to be a powerful stimulus for concentration in the cell microenvironment [23]. angiogenesis [20]. Possibly, arterialization of pre- The present results showed that the administriation existing and newly sprouted venular capillaries may of AdM increases VEGF levels in liver and lung tissues be facilitated by chronic cold exposure, as exposure but decreases VEGF levels in the brain and heart in to cold environment induces an increase in wall cold exposed rats. tension and shear stress in capillary and stimulates In conclusion, AdM treatment alters the secretion capillary angiogenesis [12]. An increase in the number of VEGF in rat liver, brain, and heart in cold exposed and density of capillaries may improve the oxygen rats, but no changes were found in lung tissue. VEGF supply to tissues during cold exposure [13-17]. may be a major regulatory factor of angiogenesis that Deveci et al. [21] suggested that cold is closely associated with cold-exposure-related exposure differentially stimulates angiogenesis in metabolic stimulation. Further studies are needed to glycolytic and oxidative muscles of rats and understand the exact role of AdM in response to cold hamsters. Kim et al. [19] showed an increase in the exposure. expression of VEGF mRNAs after prolonged cold acclimatization. Gao et al. [22] reported that cardiac The authors have no conflict of interest to report. cells were hypertrophied, the total capillary density was increased, and the diffusion area was reduced References along the capillary path from the arteriolar to venular 1. Folkman J, D’Amore PA. Blood vessel formation: what capillary portions in rats when reared at 5˜C for 68 is its molecular basis? Cell. 1996; 87:1153-5. generations. All the changes suggest that the 2. Carmeliet P, Jain RK. Angiogenesis in cancer and other hypertrophy in cardiac cells is accompanied by the diseases. Nature. 2000; 407:249-57. improvements of oxygen delivery capacity after cold 3. Crowther M, Brown NJ, Bishop ET, Lewis CE. adaptation. Microenvironmental influence on macrophage It was hypothesized that AdM plays an important regulation of angiogenesis in wounds and malignant role in the vascular remodeling by affecting the tumors. J Leukocyte Biology. 2001; 70:478-90. extracellular matrix turnover [18]. Guidolin et al. [23] 4. Risau W. Mechanisms of angiogenesis, Nature. 1997; suggested that the angiogenic actions of AdM 386:671-4. appeared independent of VEGF secretion and AdM 5. Bohem T, Folkman J, Browder T, O’Reilly MS. might act directly through up-regulation of VEGF. AdM Antiangiogenic therapy of experimental cancer does treatment did not alter the secretion of VEGF and not induce acquired drug resistance. Nature. 1997; 390: block antibodies to VEGF. Furthermore, AdM 404-7. treatment did not significantly inhibit AdM-induced in 6. Ribatti D, Conconi MT, Nussdorfer GG. Nonclassic vitro capillary-like tube formation by human umbilical endogenous regulators of angiogenesis. Pharmacol vein ECs. Rev. 2007; 59:185-205. 958 N. Cikcikoglu Yildirim, M. Yurekli

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